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Applications of BBa_K2619101

Background

In this part of experiments, we investigated the function of L7Ae, which may binds the C/D Box on our designed mRNA, and anchor the therapeutic mRNA on the inner face of exosomes. The wet lab focused on two impacts L7Ae may bring to the exosome medicine: 1. The production of exosome in exosome donor (HEK293T cells) 2. The RNA anchored in the exosome

Experiment data

1. Nanoparticle Tracking Assay

After the engreen co-transfection of L7Ae with C/D Box-nluc/nluc/empty pcDNA3.1(+), we conducted NTA to detect if the transfection may influence the exosome production, and ensure that the concentration reached 2x105 /ul, which is the recommended exosome concentration in exosome transfection (Exo-fect) in the sample ultracentrifugated. We distributed the transfected HEK293T cells in a 12-well plate as below:

We sent 50 ul of exosome isolated from each set of cells, the results were sent back and listed below:

Media:File:L7Ae-1.pdf

Figure 2: The comparison in glutamate concentration between EAAT2-transfected and pcDNA3.1(+)-transfected Neuro-2a cells, student’s t-test applied (*: p<0.05, **: p<0.01, ***: p<0.001).

The results of glutamate assay reveals that the expression of EAAT2 on cell membrane can maintain the concentration of glutamate in a relatively stable level, and significantly reduce the concentration of glutamate in the cell medium. Higher the glutamate concentration is, the more significant the EAAT2 may transport the glutamate in cells. In addition, the expression of EAAT2 will not over-transport the glutamate and make the glutamate concentration lower than the origin level.

2.WST-1 Cytotoxicity Test

To detect the cytotoxicity induced by the over-high glutamate concentration, we used WST-1 as an alternative of MTT in our test. The dehydrogenase in mitochondria can reduce the WST-1 to soluble and chromatic formazan, and a high absorbance measured indicates a low cytotoxicity induced by glutamate. The absorbance of these samples are measured at 450 nm and 690 nm as a reference wavelength. The distribution of each sample are displayed as below:

Figure 3: The absorbance (A450) measured after with reference absorbance (A690) and blank value subtracted, and its correlation to treated duration.

Figure 4: The comparison in A450- A690 between EAAT2-transfected and pcDNA3.1(+)-transfected Neuro-2a cells (after a two-hour incubation in 37 Celsius), student’s t-test applied (*: p<0.05, **: p<0.01, ***: p<0.001).

The results of WST-1 indicated that the expression of EAAT2 on cell membrane can significantly alleviate the excitotoxicity induced by glutamate. The cytotoxicity may have limited influence on cell proliferation even if the cells are cultured in inflammation-inducing (80 uM glutamate) for longer than 9 hours.

Further Application

Our primary goal is to save astrocytes from the high glutamate level at the early stage of neurodegenerative disease and strengthen the defence against the neurotoxicity. In details, we design this therapy to enhance the glutamate absorbing function by overexpression of EAAT2 on the astrocyte’s membrane. We use the mRNA-based vector for expression and protect the mRNA by exosome through the delivery process. We will further study the production, characterization, transportation, preservation and other related content of exosome therapy, and subsequently, carry out social activities related to exosome treatment to enhance our design.

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Part:BBa_K2619101:Experience